4.14.1. Antimicrobial Activity of TO@NZ Bioactive Hybrid Nanostructure

The antimicrobial activity of the TO@NZ hybrid nanostructure was estimated by the minimum inhibitory concentration (MIC) against several bacteria. The MICs of TO@NZ were studied by the broth dilution assay in Brain Heart Infusion broth (BHI, Oxoid, UK) and the selected bacteria were two lactic acid bacteria, which commonly are used as starter cultures in cheese manufacturing, namely *Lactococcus lactis* sp. *lactis* ACA-DC127 and *Streptococcus thermophilus* ST112, and 3 pathogenic strains, namely *Staphylococcus aureus* ATCC1538, *Listeria monocytogenes* NCTC10527, and *Enterococcus faecalis* EF1. All the strains were obtained from the ACA-DC Culture Collection of Food Science and Human Nutrition Department of the Agricultural University of Athens. The strains were preserved on cryobeads in cryovials of a cryopreservation system (TSC Ltd., Queensway Industrial Estate, 3–4 Arkwright Way, Scunthorpe DN16 1AL UK) at −80 ◦C, and overnight cultures were grown in BHI broth at 30 ◦C for *L. Lactis* ACA-DC127, and 37 ◦C for *S. thermophilus* ST112 and all pathogenic strains. For the broth dilution assay, five BHI broths with TO@NZ concentrations of 0.01, 0.025, 0.05, 0.075, and 0.1% (*w*/*v*) were made. The selected bacteria were inoculated into BHI broths, in a final concentration of approximately 10<sup>6</sup> cfu/mL in the presence of the above different concentrations of TO@NZ hybrid nanostructure, and incubation was followed for 48 h at the appropriate temperature for each strain. Growth was assessed by measuring the absorbance at 625 mM and agar plating after incubation for 48 h at 30 ◦C for the *L. lactis* sp. *lactis* AC A-DC127 strain and at 37◦ for the other bacteria to determine viable cfu/mL. The MIC values were read as the lowest concentrations of

TO@NZ at which bacterial growth was completely inhibited. In addition, to determine the minimal bactericidal concentration (MBC), the dilution representing the MIC and at least two of the more concentrated TO@NZ dilutions were plated and enumerated to determine viable cfu/mL. The MBC value was estimated as the lowest concentration that demonstrated a reduction (such as 99.9%) in cfu/mL of the strain tested.
